Breathing is a continuous and rhythmic behavior that is under exquisite control by the nervous system. The brain regulates breathing not only to maintain homeostasis across different conditions, but also to support other diverse elements of fundamental human behavior. Breathing is controlled to express emotions, e.g., laughing, crying, and is modulated across various emotional states, e.g., hyperventilation during fear, anxiety. Voluntary or cognitive control of breathing is essential for communication, to generate sounds for speech and phonation, and even to influence the emotional and physiological state, e.g., during meditation or breathing exercises. How is the brain wired to enable such extraordinary levels of control? Addressing this issue is critical since disturbances to the emotive and volitional control of breathing have a significant health impact, as anxiety disorders and genetic disorders such as Rett syndrome manifest abnormal breathing patterns. The key to understanding how the brain gives rise to such respiratory dysfunction and for developing treatments is to uncover the underlying neural circuitry and, with this information, determine the functional contribution of neuronal subtypes within the breathing control circuit. While the principal brainstem regions responsible for generating the breathing rhythm, the preBtzinger Complex (preBtC) and the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) are known, the higher brain centers that provide input to modulate breathing have not been well characterized. This proposal focuses on these two critical brainstem respiratory centers and aims to examine their connectivity with higher brain regions, e.g., amygdala, hypothalamus, or cortex, that provide signals underlying the emotive and volitional control for breathing. To delineate this neural circuit, I will exploit a recently developed genetic viral strategy to target molecularly defined neurons in the preBtC or RTN/pFRG. Experiments proposed in Aim 1 will identify the higher brain regions that provide input to the preBtC or RTN/pFRG by infecting specific subpopulations of these neurons with a virus that can be transported retrogradely across the synapse. The virus will label presynaptic neurons with a genetically encoded fluorescent protein, allowing identification and characterization of all input neurons that send direct afferent projections to the respiratory control areas. The goal of Aim 2 is to determine how preBtC or RTN/pFRG neurons transmit information to higher brain regions. Subpopulations of preBtC or RTN/pFRG neurons will be infected with a virus expressing a genetically encoded fluorescent protein to enable visualization of the efferent projections and their target neurons. This proposal will use state-of-the-art technology to map the connectivity of the regions that generate the breathing rhythm with higher brain regions, which will reveal fundamental insight into the emotive and volitional control of breathing and uncover mechanisms underlying human breathing disorders.